Epitopes Identification Of Structural Protein Of Chicken Anemia Virus And Construction Of CAV Infectious Clone

To research the epitopes of VP1 protein of chicken anemia virus, vp1 gene was divided into six overlapping fragments based on the analysis of antigenic index using sofeware Lasergene. All the fragments were expressed in E.coli subsequently. Six monoclonal antibodies(MAbs) were developed using the purified VP1 protein as immunogen. The reactivity of different fragments expressed in E.coli was probed with 6 monoclonal antibodies by Western blot. According to the results of Western blot, three antigenic epitope sites were identified and they were located in 219～274 amino acid(aa), 324～369 aa, and 275～302 aa, respectively.To map the antigenic structure of VP1 protien in detail, vp1-gene-targeted library was constructed. After 3 rounds of panning with polyclonal antibodies, 9 positive clones were obtained. The results of sequence alignment showed that five of them shared a consensus sequence T³⁰⁹YMSFATLTALGAQWSFPPGQRSVSRRSFNHHKARG³⁴⁴, and the other four of them shared a consensus sequence M¹⁹⁶FGGWHLFRHIETRFQLLA²¹⁴. These positive phages were probed with 6 MAbs by Western blot and the results showed that 4 of 6 MAbs reacted with clone P12, so the epitope recognized by MAb 2F3 was located in 219～253 aa. To map the precise antigenic epitopes of the MAb 4D5 and MAb 2F3, a series of oligonucleotides were synthesized. The synthesized-oligonucleotides were inserted into the pET32a vector after annealing and expressed in host E.coli BL21(DE3). On the basis of reactivity of MAbs to the expressed fusion peptides in Western blot, the epitope recognized by MAb 2F3 was D²⁴⁵HQNRWRKG²⁵³ and the epitope recognized by MAb 4D5 was W³⁵³HTLVPLGTE³⁶².The analysis of software showed that VP2 protein was of good antigenic index. To map the epitopes of VP2 protein of chicken anemia virus(CAV), VP2 was expressed as a fusion protein in Escherichia coli BL21(DE3). Female BALB/c mice were immunized with purified recombinant VP2 produced in E. coli BL21(DE3) and seven VP2-specific monoclonal antibodies were developed. The VP2 protein was dissected into 21 overlapping fragments, expressed as fusion products in E. coli, and used for epitope mapping by pepscan analysis. Three antigenic epitopes were identified by ELISA and Western blot assays, which were G²¹QPGPSGAAQGQVISN³⁶, L¹¹¹EDRSTQASLEEAILR¹²⁶ and E¹²¹EAILRPLRVQGKRAK¹³⁶, respectively. According to the reactivity of MAbs with neighbouring fragments, it was demonstrated that G¹⁶AAQ²⁰, Q¹¹⁷ASL¹²⁰ and P¹²⁷LRV¹³⁰ were main functional region of each epitope. The three epitope peptides were purified and used to immunize BALB/c mice. The Western blot analysis showed that all the fusion peptides induced VP2 protein specific antibodies.To research how vp3 gene affects the viral replication and virulence, infectious molecular clone of CAV and two vp3 mutations were constructed. In the present study, an infectious clone of CAV was constructed by ligating two copies of the complete CAV genome in tandem into the pBluescriptâ…¡SK vector and was shown to be infectious in vitro when transfected into MDCC-MSB1 cells. Ten chicks were intramuscularly inoculated with 100μg of the cloned CAV plasmid and inoculated chicks developed the symptoms resembling that induced by intramuscular inoculation with CAV live virus stock. To further examine the role of VP3 in viral replication and virulence, the infectious molecular clone of CAV was subjected to site-directed mutagenesis and resulted in the deletion of VP3 and NLS2 of VP3. Two mutants of infectious molecular clone pBluem2CAWP3^-, and pBluem2CAVNLS2^-were constructed, respectively. The results of transfection with two mutated infectious clones showed that CAV could not replicate when the vp3 gene was deleted completely, but virus could replicate with deletion of NLS2 in vitro. It showed the same results as in vitro when the two mutants of infectious clone were intramuscularly inoculated into chicks. The virus could not replicate in vivo when the vp3 gene was deleted completely, but virus with deletion of NLS2 of vp3 could replicate in vivo and consequently caused the increase of the thymus glands. The construction of CAV infectious clone laid a good foundation for purification of CAV and development of vaccine. The research on the two mutated virus is useful for further study the function of vp3 gene and construction of low pathogenicity virus.